Critical sized bone defects caused by injury, disease or congenital malformations, remain a challenging problem in orthopedic medicine. Current options to restore full function to such bone defects are limited due to slow rates of regeneration of native bone tissue, second site morbidity, poor mechanical stability and lack of integration with surrounding tissues depending on the mode of clinical repair utilized. New options to accelerate the rate and extent of new bone formation, as well as integration to surrounding tissues are needed to overcome current limitations. In this competitive renewal application, a novel silk protein matrix will be bioengineered to optimize these goals to achieve large defect bone regeneration. The proposed studies build off of the results from the current grant that demonstrated the unique and useful attributes of a silk fibroin protein 3D porous matrix for in vitro and in vivo bone regeneration. In the proposed research, our goal is to accelerate the rate and extent of bone formation and integration across the defect through the combined delivery of BMP-2 and VEGF in the 3D protein matrices, and to incorporate bioengineered peptide adhesives to promote interactions with adjacent parent bone. These enhanced, degradable and biocompatible 3D porous silk matrices functionalized with growth factors and adhesion capabilities will be studied in a rat critical sized femur defect model to optimize their design. Subsequent to optimization, in the final aim of the study, the implants will be assessed in a critical-size goat femur defect model. Our goal is to conclude the study with an optimized design for these new 3D porous protein matrices in order to pursue human clinical trials. Outcome assessments for the three aims will be based on mineral density, homogeneity of mineral distribution and mechanical integrity of the repairs in the small and then large animal critical sized defects. To achieve the goals, an interdisciplinary team of investigators has been assembled to address the challenges with expertise in biomaterial matrix design, stem cell biology, biomechanics, imaging and veterinary medicine.